Surface concatemerization of templates

ABSTRACT

Presented herein are methods and compositions for concatenating template strands during the bridge amplification process. The methods are useful for surface amplification at improved densities. The methods and compositions provided herein enable creation of clusters which are brighter, but at the same densities as currently achieved using standard cluster amplification.

BACKGROUND

The task of cataloguing human genetic variation and correlating thisvariation with susceptibility to disease is daunting and expensive. Adrastic reduction in this cost is imperative for advancing theunderstanding of health and disease. A reduction in sequencing costswill require a number of technical advances in the field. Technicaladvances that could reduce the cost of genome analysis include: (1)library generation; (2) highly-parallel clonal amplification andanalysis; (3) development of robust cycle sequencing biochemistry; (4)development of ultrafast imaging technology; and (5) development ofalgorithms for sequence assembly from short reads.

The creation of clonal amplifications in a highly-parallel manner isimportant for cost-effective sequencing. Sequencing is generallyperformed on clonal populations of DNA molecules traditionally preparedfrom plasmids grown from picking individual bacterial colonies. In thehuman genome project, each clone was individually picked, grown-up, andthe DNA extracted or amplified out of the clone. In recent years, therehave been a number of innovations to enable highly-parallelized analysisof DNA clones particularly using array-based approaches. In the simplestapproach, the library can be analyzed at the single molecule level whichby its very nature is clonal. The major advantage of single moleculesequencing is that cyclic sequencing can occur asynchronously since eachmolecule is read out individually. In contrast, analysis of clonalamplifications requires near quantitative completion of each sequencingcycle, otherwise background noise progressively grows with each ensuingcycle severely limiting read length. As such, clonal analysis places abigger burden on the robustness of the sequencing biochemistry and maypotentially limit read lengths.

Thus, there exists a need to develop methods to improve genomicsanalysis and provide more cost effective methods for sequence analysis.The present invention satisfies this need and provides relatedadvantages as well.

BRIEF SUMMARY

The methods and compositions provided herein enable surfaceamplification at improved densities. Described herein are methods forconcatenating template strands during the bridge amplification process,such that each flowcell surface primer can end up with a multiple copiesof the template strand extended onto it. The methods and compositionsprovided herein enable creation of clusters which are brighter, but atthe same densities as currently achieved using standard clusteramplification. Brighter clusters may have a number of advantages, forexample, better quality of reads, support for longer read lengths,faster scan times for sequencing, and increased system robustness.

Presented herein is a method of preparing immobilized templates for anucleic acid sequencing reaction comprising: (a) providing a solidsupport having a forward and reverse amplification primer immobilizedthereon; (b) providing a target nucleic acid, wherein the target nucleicacid comprises: (i) a first region of known sequence complementary tothe forward amplification primer; (ii) a first template region; (iii) asecond region of known sequence substantially identical to the reverseamplification primer, wherein the first template region is between thefirst region of known sequence and the second region of known sequence;and (iv) a third region of known sequence complementary to the forwardamplification primer, wherein the first template region and the secondregion of known sequence are between the first region of known sequenceand the third region of known sequence; (c) applying the target nucleicacid to the solid support under conditions suitable for hybridizationwhereby the first region of known sequence hybridizes to the forwardamplification primer; and (d) extending the hybridized forwardamplification primer to generate an immobilized template comprising acomplementary copy of the target nucleic acid.

In some embodiments, the method can further comprise: (e) denaturing thetarget nucleic acid from the immobilized template; (f) hybridizing theimmobilized template to the reverse amplification primer, whereby acomplementary copy of the second flanking sequence hybridizes to thereverse amplification primer; and (g) extending the hybridized reverseamplification primer to generate a second immobilized strand comprisingthe first template region positioned between the first region of knownsequence and the second region of known sequence.

In some embodiments, the method can further comprise: (h) denaturing thesecond immobilized strand from the first immobilized template; (i)hybridizing the second immobilized strand to the first immobilizedtemplate, whereby the first region of known sequence of the secondimmobilized strand hybridizes to a complementary copy of the firstregion of known sequence in the immobilized template; (j) extending a 3′OH of the first region of known sequence to generate a concatemer of thesecond immobilized strand; and (k) extending a 3′ OH of thecomplementary copy of the first region of known sequence to generate aconcatemer of the first immobilized template.

In some embodiments, the method can further comprise: (1) denaturing theconcatemers and repeating steps (i), (j) and (k) to generate furtherconcatemers of each strand.

In some aspects of the above embodiments, the forward amplificationprimer can comprise a forward complementarity region, said forwardcomplementarity region having complementarity to a reversecomplementarity region of the reverse amplification primer.

In some aspects, the first complementarity region is positioned directly5′ of a region having complementarity to the first region of knownsequence of the target nucleic acid.

In some aspects, the reverse complementarity region is positioneddirectly 5′ of a region having sequence substantially identical to thesecond region of known sequence of the target nucleic acid.

In some aspects, the forward complementarity region and secondcomplementarity region are configured such that repeated cycles ofhybridization and extension produces a concatemer of the firstimmobilized strand, each copy in the concatemer separated from the nextby sequence from the forward complementarity region.

In some aspects, the forward complementarity region and secondcomplementarity region are configured such that repeated cycles ofhybridization and extension produces a concatemer of the secondimmobilized strand, each copy in the concatemer separated from the nextby sequence from the reverse complementarity region.

In some aspects, the concatemers comprises multiple copies of saidtarget nucleic acid molecule of at least 10, 20, 50, 100, 200, 500,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or at least 10,000copies.

In some aspects, the solid support is planar. In some aspects, the solidsupport comprises microwells.

In some aspects, the target nucleic acid has a length of at least 10,20, 50, 100, 200 or at least 500 nucleotides.

In some aspects, the target nucleic acid further comprises a fourthregion of known sequence substantially identical to the reverseamplification primer, wherein the first region of known sequence, thefirst template region and the second region of known sequence arebetween the third region of known sequence and the fourth region ofknown sequence.

In some aspects, the forward amplification primer comprises anon-nucleotide chemical linker moiety positioned to prevent copying ofany nucleotides that are 5′ of the non-nucleotide chemical linkermoiety. In some aspects, the reverse amplification primer comprises anon-nucleotide chemical linker moiety positioned to prevent copying ofany nucleotides that are 5′ of the non-nucleotide chemical linkermoiety. In aspects of any of the above-described embodiments, thenon-nucleotide chemical linker moiety can comprises a diol moiety. Insome aspects, the non-nucleotide chemical linker moiety comprises anon-nucleotide linker tethering the primer to the solid support.

In some embodiments, the method can further comprise sequencing thetarget nucleic acid. For example, in some aspects, sequencing the targetnucleic acid comprises: hybridizing one or more sequencing primers tothe first immobilized template or the second immobilized strand;extending the sequencing primers by incorporating one or more labelednucleotides into the nascent strand; and detecting the labelednucleotides, thereby obtaining sequence information about the targetnucleic acid.

In some embodiments, the target nucleic acid is prepared by amplifyingwith a pair of primers configured with a binding moiety whereby onestrand of an amplification product generated using the pair of primerscan be separated from the opposite strand. In some aspects, the bindingmoiety comprises biotin and can be separated by binding to a solidsupport comprising streptavidin.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the description and drawings, and fromthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing solid phase amplification according to oneembodiment.

FIG. 2 is a schematic showing alternative library constructs for use insolid phase amplification.

FIG. 3 is a schematic showing solid phase amplification according to oneembodiment.

FIG. 4A is a fluorescence microscope image of amplification clustersaccording standard protocols compared to one embodiment presentedherein.

FIG. 4B is a plot of number of clusters versus the SYBR signal intensityfrom the flowcells described in FIG. 4A.

FIG. 5 is a set of fluorescence images of fluorescent nucleotides C, Tand G incorporated into clusters amplified according to standardprotocols or according to one embodiment presented herein.

FIG. 6 shows fluorescence images (top) and histograms (bottom) ofclusters amplified according to standard protocols or according to oneembodiment presented herein.

FIG. 7 shows a comparison of cluster intensity and size for variousamplification protocols.

FIG. 8 shows a comparison of signal intensity over 100 cycles in surfaceconcatemer-containing clusters versus standard clusters.

DETAILED DESCRIPTION

Standard technology for surface amplification of nucleic acid templates(“clustering”) has enabled very high densities (500-600 k/mm²) on atypical flowcell. However, there remains a need for improvedmethodologies for surface amplification of nucleic acid templates. Themethods and compositions provided herein enable surface amplification atimproved densities. Described herein are methods for concatenatingtemplate strands during the bridge amplification process, such that eachflowcell surface primer can end up with a multiple copies of thetemplate strand extended onto it. The methods and compositions providedherein enable creation of clusters which are brighter, but at the samedensities as currently achieved using standard cluster amplification.Brighter clusters may have a number of advantages, for example, betterquality of reads, support for longer read lengths, faster scan times forsequencing, and increased system robustness.

One embodiment is illustrated in FIG. 1, which sets forth a method offorming concatemers of a target nucleic acid. As shown in the boxedportion of FIG. 1, a solid support is provided having immobilizedthereon a plurality of forward and reverse amplification primers,designated P5 and P7 in the figure. A target nucleic acid is alsoprovided, having a first region of known sequence (P5′), complementaryto P5 primer, a second region of known sequence substantially identicalto the reverse amplification primer (P7) and a third region of knownsequence (P5′), complementary to P5 primer. A first template region ispositioned between the first (P5′) and second (P7) regions of knownsequence. The target nucleic acid is applied to the solid support underconditions suitable for hybridization such that the P5′ sequencehybridizes to the P5 sequence of the immobilized primer. The primer isextended under conditions favorable to extension, for example in thepresence of a DNA polymerase and dNTPs. The resulting extension productis an immobilized template comprising a complementary copy of the targetnucleic acid, as illustrated in FIG. 1 after the 1^(st) extension.

Further rounds of bridge amplification can include denaturing the targetnucleic acid from the immobilized template and allowing the immobilizedtemplate to hybridize to the reverse amplification primer. In theexample shown in FIG. 1, the complementary copy of the second knownsequence (P7′) hybridizes to the reverse primer (P7) and is extendedunder extension conditions described above. The resulting extensionproduct is a second immobilized strand comprising the first templateregion positioned between the first region of known sequence and thesecond region of known sequence, as illustrated in FIG. 1 after the2^(nd) extension. Subsequent cycles of denaturation, hybridization andextension allow the process to continue such that the second immobilizedstrand can hybridize to the first immobilized template and one or both3′ OH of the second immobilized strand and/or the first immobilizedtemplate are extended to generate further extension products. Thefurther extension products that result from these additional cycles areconcatemers of one or both of the second immobilized strand and/or thefirst immobilized template, as illustrated in FIG. 1 after the nextcycles. The concatemers can include 2 or more copies of the templateregion (or complement thereof) positioned in between the knownsequences. In some embodiments, the concatemers can include 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 50, 100, 200, 500, 1000, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000 or more than 10,000 copies of the template region(or complement thereof) positioned in between the known sequences. Afterrounds of amplification, a sequencing primer in solution can behybridized to a region of known sequence and extended. The sequencingprimer will hybridize to multiple locations on the same immobilizedstrand and/or immobilized template. Multiple sequencing reactions cantherefore take place on the same strand, resulting in a signal that isgreatly amplified over previous methods, where only one signal perstrand is generated.

As used herein, the term “concatemer” refer to a long continuous nucleicacid molecule that contains multiple copies of the same sequences linkedin series. The multiple copies may be separated by other sequence, forexample, by known sequence that flanks the concatemerized sequence onthe 3′ and/or the 5′ end. Any number of copies of flanking sequence maybe interspersed between the concatemerized sequence. In someembodiments, the concatemers can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,50, 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000or more than 10,000 copies of a template region (or complement thereof)on a single concatemer. In some embodiments, the concatemers cancomprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 500, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000 or more than 10,000 copies ofknown sequence, positioned in a repeating manner between copies of theknown sequences. Further exemplification of this embodiment is set forthin Example 1 below.

FIG. 2 illustrates an alternate embodiment, where a strand complementaryto the target nucleic acid is provided. This may naturally result inembodiments where the target nucleic acid is provided in double-strandedform. As shown in FIG. 2, the opposite strand hybridizes to the reverseamplification primer and is extended. However, in contrast to theembodiment illustrated in FIG. 1, a single copy of the target nucleicacid is generated. Further cycles of amplification will not generateconcatemers, as shown in FIG. 2. To avoid this result, alternativelibrary constructs can be generated that comprise both the first andsecond known regions on both the 3′ end and the 5′ end of thedouble-stranded construct, as illustrated in FIG. 2. Amplification ofeither the top strand or the bottom strand will generate concatemers, ina manner similar to that shown in FIG. 1.

FIG. 3 illustrates another embodiment, wherein the forward and reverseamplification primers comprise additional sequence positioned 5′ of thesequence that hybridizes to the known sequence. In some embodiments, theadditional sequence of the forward primer is complementary to theadditional sequence of the reverse primer. For example, as illustratedin FIG. 3, the surface primers comprise an oligo dA and oligo dTsequence positioned 5′ of the P5 and P7 sequences (designated 20T-P7 and20A-P5). Thus, after a 1^(st) extension cycle, the immobilized templatecomprises the complementarity sequence (20mer A) immobilized at the 5′end of the template, as shown in FIG. 1 after the 1^(st) extension.After a subsequent extension, the resulting copy comprises thecomplement of the complementarity sequence (20mer T) immobilized at the5′ end of the copied strand and again at the 3′ end of the extensionproduct. As further rounds of amplification take place, thecomplementarity sequences are incorporated in tandem, flanking the knownsequences, for example P5 and P7, as illustrated in FIG. 3. Furtherexemplification of this embodiment is set forth in Example 2 below.

Cluster Amplification

Described herein are methods for concatenating template strands duringthe bridge amplification process. Bridge amplification is also referredherein as “cluster amplification.” In some embodiments, the immobilizedDNA fragments are amplified using cluster amplification methodologies asexemplified by the disclosures of U.S. Pat. Nos. 7,985,565 and7,115,400, the contents of each of which is incorporated herein byreference in its entirety. The incorporated materials of U.S. Pat. Nos.7,985,565 and 7,115,400 describe methods of solid-phase nucleic acidamplification which allow amplification products to be immobilized on asolid support in order to form arrays comprised of clusters or“colonies” of immobilized nucleic acid molecules. Each cluster or colonyon such an array is formed from a plurality of identical immobilizedpolynucleotide strands and a plurality of identical immobilizedcomplementary polynucleotide strands. The arrays so-formed are generallyreferred to herein as “clustered arrays”. The products of solid-phaseamplification reactions such as those described in U.S. Pat. Nos.7,985,565 and 7,115,400 are so-called “bridged” structures formed byannealing of pairs of immobilized polynucleotide strands and immobilizedcomplementary strands, both strands being immobilized on the solidsupport at the 5′ end, preferably via a covalent attachment. Clusteramplification methodologies are examples of methods wherein animmobilized nucleic acid template is used to produce immobilizedamplicons. Other suitable methodologies can also be used to produceimmobilized amplicons from immobilized DNA fragments produced accordingto the methods provided herein. For example one or more clusters orcolonies can be formed via solid-phase PCR whether one or both primersof each pair of amplification primers are immobilized.

In other embodiments, the immobilized DNA fragments are amplified insolution. For example, in some embodiments, the immobilized DNAfragments are cleaved or otherwise liberated from the solid support andamplification primers are then hybridized in solution to the liberatedmolecules. In other embodiments, amplification primers are hybridized tothe immobilized DNA fragments for one or more initial amplificationsteps, followed by subsequent amplification steps in solution. Thus, insome embodiments an immobilized nucleic acid template can be used toproduce solution-phase amplicons.

It will be appreciated that any of the amplification methodologiesdescribed herein or generally known in the art can be utilized withuniversal or target-specific primers to amplify immobilized DNAfragments. Suitable methods for amplification include, but are notlimited to, the polymerase chain reaction (PCR), strand displacementamplification (SDA), transcription mediated amplification (TMA) andnucleic acid sequence based amplification (NASBA), as described in U.S.Pat. No. 8,003,354, which is incorporated herein by reference in itsentirety. The above amplification methods can be employed to amplify oneor more nucleic acids of interest. For example, PCR, including multiplexPCR, SDA, TMA, NASBA and the like can be utilized to amplify immobilizedDNA fragments. In some embodiments, primers directed specifically to thenucleic acid of interest are included in the amplification reaction.

Other suitable methods for amplification of nucleic acids can includeoligonucleotide extension and ligation, rolling circle amplification(RCA) (Lizardi et al., Nat. Genet. 19:225-232 (1998), which isincorporated herein by reference) and oligonucleotide ligation assay(OLA) (See generally U.S. Pat. Nos. 7,582,420, 5,185,243, 5,679,524 and5,573,907; EP 0 320 308 B1; EP 0 336 731 B1; EP 0 439 182 B1; WO90/01069; WO 89/12696; and WO 89/09835, all of which are incorporated byreference) technologies. It will be appreciated that these amplificationmethodologies can be designed to amplify immobilized DNA fragments. Forexample, in some embodiments, the amplification method can includeligation probe amplification or oligonucleotide ligation assay (OLA)reactions that contain primers directed specifically to the nucleic acidof interest. In some embodiments, the amplification method can include aprimer extension-ligation reaction that contains primers directedspecifically to the nucleic acid of interest. As a non-limiting exampleof primer extension and ligation primers that can be specificallydesigned to amplify a nucleic acid of interest, the amplification caninclude primers used for the GoldenGate assay (Illumina, Inc., SanDiego, Calif.) as exemplified by U.S. Pat. Nos. 7,582,420 and 7,611,869,each of which is incorporated herein by reference in its entirety.

Exemplary isothermal amplification methods that can be used in a methodof the present disclosure include, but are not limited to, MultipleDisplacement Amplification (MDA) as exemplified by, for example Dean etal., Proc. Natl. Acad. Sci. USA 99:5261-66 (2002) or isothermal stranddisplacement nucleic acid amplification exemplified by, for example U.S.Pat. No. 6,214,587, each of which is incorporated herein by reference inits entirety. Other non-PCR-based methods that can be used in thepresent disclosure include, for example, strand displacementamplification (SDA) which is described in, for example Walker et al.,Molecular Methods for Virus Detection, Academic Press, Inc., 1995; U.S.Pat. Nos. 5,455,166, and 5,130,238, and Walker et al., Nucl. Acids Res.20:1691-96 (1992) or hyperbranched strand displacement amplificationwhich is described in, for example Lage et al., Genome Research13:294-307 (2003), each of which is incorporated herein by reference inits entirety. Isothermal amplification methods can be used with thestrand-displacing Phi 29 polymerase or Bst DNA polymerase largefragment, 5′->3′ exo- for random primer amplification of genomic DNA.The use of these polymerases takes advantage of their high processivityand strand displacing activity. High processivity allows the polymerasesto produce fragments that are 10-20 kb in length. As set forth above,smaller fragments can be produced under isothermal conditions usingpolymerases having low processivity and strand-displacing activity suchas Klenow polymerase. Additional description of amplification reactions,conditions and components are set forth in detail in the disclosure ofU.S. Pat. No. 7,670,810, which is incorporated herein by reference inits entirety.

Another nucleic acid amplification method that is useful in the presentdisclosure is Tagged PCR which uses a population of two-domain primershaving a constant 5′ region followed by a random 3′ region as described,for example, in Grothues et al. Nucleic Acids Res. 21(5):1321-2 (1993),incorporated herein by reference in its entirety. The first rounds ofamplification are carried out to allow a multitude of initiations onheat denatured DNA based on individual hybridization from therandomly-synthesized 3′ region. Due to the nature of the 3′ region, thesites of initiation are contemplated to be random throughout the genome.Thereafter, the unbound primers can be removed and further replicationcan take place using primers complementary to the constant 5′ region.

Sequencing Methods

The methods described herein can be used in conjunction with a varietyof nucleic acid sequencing techniques. Particularly applicabletechniques are those wherein nucleic acids are attached at fixedlocations in an array such that their relative positions do not changeand wherein the array is repeatedly imaged. Embodiments in which imagesare obtained in different color channels, for example, coinciding withdifferent labels used to distinguish one nucleotide base type fromanother are particularly applicable. In some embodiments, the process todetermine the nucleotide sequence of a target nucleic acid can be anautomated process. Preferred embodiments include sequencing-by-synthesis(“SBS”) techniques.

SBS techniques generally involve the enzymatic extension of a nascentnucleic acid strand through the iterative addition of nucleotidesagainst a template strand. In traditional methods of SBS, a singlenucleotide monomer may be provided to a target nucleotide in thepresence of a polymerase in each delivery. However, in the methodsdescribed herein, more than one type of nucleotide monomer can beprovided to a target nucleic acid in the presence of a polymerase in adelivery.

SBS can utilize nucleotide monomers that have a terminator moiety orthose that lack any terminator moieties. Methods utilizing nucleotidemonomers lacking terminators include, for example, pyrosequencing andsequencing using γ-phosphate-labeled nucleotides, as set forth infurther detail below. In methods using nucleotide monomers lackingterminators, the number of nucleotides added in each cycle is generallyvariable and dependent upon the template sequence and the mode ofnucleotide delivery. For SBS techniques that utilize nucleotide monomershaving a terminator moiety, the terminator can be effectivelyirreversible under the sequencing conditions used as is the case fortraditional Sanger sequencing which utilizes dideoxynucleotides, or theterminator can be reversible as is the case for sequencing methodsdeveloped by Solexa (now Illumina, Inc.).

SBS techniques can utilize nucleotide monomers that have a label moietyor those that lack a label moiety. Accordingly, incorporation events canbe detected based on a characteristic of the label, such as fluorescenceof the label; a characteristic of the nucleotide monomer such asmolecular weight or charge; a byproduct of incorporation of thenucleotide, such as release of pyrophosphate; or the like. Inembodiments, where two or more different nucleotides are present in asequencing reagent, the different nucleotides can be distinguishablefrom each other, or alternatively, the two or more different labels canbe the indistinguishable under the detection techniques being used. Forexample, the different nucleotides present in a sequencing reagent canhave different labels and they can be distinguished using appropriateoptics as exemplified by the sequencing methods developed by Solexa (nowIllumina, Inc.).

Preferred embodiments include pyrosequencing techniques. Pyrosequencingdetects the release of inorganic pyrophosphate (PPi) as particularnucleotides are incorporated into the nascent strand (Ronaghi, M.,Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996)“Real-time DNA sequencing using detection of pyrophosphate release.”Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) “Pyrosequencingsheds light on DNA sequencing.” Genome Res. 11(1), 3-11; Ronaghi, M.,Uhlen, M. and Nyren, P. (1998) “A sequencing method based on real-timepyrophosphate.” Science 281(5375), 363; U.S. Pat. No. 6,210,891; U.S.Pat. No. 6,258,568 and U.S. Pat. No. 6,274,320, the disclosures of whichare incorporated herein by reference in their entireties). Inpyrosequencing, released PPi can be detected by being immediatelyconverted to adenosine triphosphate (ATP) by ATP sulfurylase, and thelevel of ATP generated is detected via luciferase-produced photons. Thenucleic acids to be sequenced can be attached to features in an arrayand the array can be imaged to capture the chemiluminscent signals thatare produced due to incorporation of a nucleotides at the features ofthe array. An image can be obtained after the array is treated with aparticular nucleotide type (e.g. A, T, C or G). Images obtained afteraddition of each nucleotide type will differ with regard to whichfeatures in the array are detected. These differences in the imagereflect the different sequence content of the features on the array.However, the relative locations of each feature will remain unchanged inthe images. The images can be stored, processed and analyzed using themethods set forth herein. For example, images obtained after treatmentof the array with each different nucleotide type can be handled in thesame way as exemplified herein for images obtained from differentdetection channels for reversible terminator-based sequencing methods.

In another exemplary type of SBS, cycle sequencing is accomplished bystepwise addition of reversible terminator nucleotides containing, forexample, a cleavable or photobleachable dye label as described, forexample, in WO 04/018497 and U.S. Pat. No. 7,057,026, the disclosures ofwhich are incorporated herein by reference. This approach is beingcommercialized by Solexa (now Illumina Inc.), and is also described inWO 91/06678 and WO 07/123,744, each of which is incorporated herein byreference. The availability of fluorescently-labeled terminators inwhich both the termination can be reversed and the fluorescent labelcleaved facilitates efficient cyclic reversible termination (CRT)sequencing. Polymerases can also be co-engineered to efficientlyincorporate and extend from these modified nucleotides.

Preferably in reversible terminator-based sequencing embodiments, thelabels do not substantially inhibit extension under SBS reactionconditions. However, the detection labels can be removable, for example,by cleavage or degradation. Images can be captured followingincorporation of labels into arrayed nucleic acid features. Inparticular embodiments, each cycle involves simultaneous delivery offour different nucleotide types to the array and each nucleotide typehas a spectrally distinct label. Four images can then be obtained, eachusing a detection channel that is selective for one of the fourdifferent labels. Alternatively, different nucleotide types can be addedsequentially and an image of the array can be obtained between eachaddition step. In such embodiments each image will show nucleic acidfeatures that have incorporated nucleotides of a particular type.Different features will be present or absent in the different images duethe different sequence content of each feature. However, the relativeposition of the features will remain unchanged in the images. Imagesobtained from such reversible terminator-SBS methods can be stored,processed and analyzed as set forth herein. Following the image capturestep, labels can be removed and reversible terminator moieties can beremoved for subsequent cycles of nucleotide addition and detection.Removal of the labels after they have been detected in a particularcycle and prior to a subsequent cycle can provide the advantage ofreducing background signal and crosstalk between cycles. Examples ofuseful labels and removal methods are set forth below.

In particular embodiments some or all of the nucleotide monomers caninclude reversible terminators. In such embodiments, reversibleterminators/cleavable fluors can include fluor linked to the ribosemoiety via a 3′ ester linkage (Metzker, Genome Res. 15:1767-1776 (2005),which is incorporated herein by reference). Other approaches haveseparated the terminator chemistry from the cleavage of the fluorescencelabel (Ruparel et al., Proc Natl Acad Sci USA 102: 5932-7 (2005), whichis incorporated herein by reference in its entirety). Ruparel et aldescribed the development of reversible terminators that used a small 3′allyl group to block extension, but could easily be deblocked by a shorttreatment with a palladium catalyst. The fluorophore was attached to thebase via a photocleavable linker that could easily be cleaved by a 30second exposure to long wavelength UV light. Thus, either disulfidereduction or photocleavage can be used as a cleavable linker. Anotherapproach to reversible termination is the use of natural terminationthat ensues after placement of a bulky dye on a dNTP. The presence of acharged bulky dye on the dNTP can act as an effective terminator throughsteric and/or electrostatic hindrance. The presence of one incorporationevent prevents further incorporations unless the dye is removed.Cleavage of the dye removes the fluor and effectively reverses thetermination. Examples of modified nucleotides are also described in U.S.Pat. No. 7,427,673, and U.S. Pat. No. 7,057,026, the disclosures ofwhich are incorporated herein by reference in their entireties.

Additional exemplary SBS systems and methods which can be utilized withthe methods and systems described herein are described in U.S. PatentApplication Publication No. 2007/0166705, U.S. Patent ApplicationPublication No. 2006/0188901, U.S. Pat. No. 7,057,026, U.S. PatentApplication Publication No. 2006/0240439, U.S. Patent ApplicationPublication No. 2006/0281109, PCT Publication No. WO 05/065814, U.S.Patent Application Publication No. 2005/0100900, PCT Publication No. WO06/064199, PCT Publication No. WO 07/010,251, U.S. Patent ApplicationPublication No. 2012/0270305 and U.S. Patent Application Publication No.2013/0260372, the disclosures of which are incorporated herein byreference in their entireties.

Some embodiments can utilize detection of four different nucleotidesusing fewer than four different labels. For example, SBS can beperformed utilizing methods and systems described in the incorporatedmaterials of U.S. Patent Application Publication No. 2013/0079232. As afirst example, a pair of nucleotide types can be detected at the samewavelength, but distinguished based on a difference in intensity for onemember of the pair compared to the other, or based on a change to onemember of the pair (e.g. via chemical modification, photochemicalmodification or physical modification) that causes apparent signal toappear or disappear compared to the signal detected for the other memberof the pair. As a second example, three of four different nucleotidetypes can be detected under particular conditions while a fourthnucleotide type lacks a label that is detectable under those conditions,or is minimally detected under those conditions (e.g., minimal detectiondue to background fluorescence, etc). Incorporation of the first threenucleotide types into a nucleic acid can be determined based on presenceof their respective signals and incorporation of the fourth nucleotidetype into the nucleic acid can be determined based on absence or minimaldetection of any signal. As a third example, one nucleotide type caninclude label(s) that are detected in two different channels, whereasother nucleotide types are detected in no more than one of the channels.The aforementioned three exemplary configurations are not consideredmutually exclusive and can be used in various combinations. An exemplaryembodiment that combines all three examples, is a fluorescent-based SBSmethod that uses a first nucleotide type that is detected in a firstchannel (e.g. dATP having a label that is detected in the first channelwhen excited by a first excitation wavelength), a second nucleotide typethat is detected in a second channel (e.g. dCTP having a label that isdetected in the second channel when excited by a second excitationwavelength), a third nucleotide type that is detected in both the firstand the second channel (e.g. dTTP having at least one label that isdetected in both channels when excited by the first and/or secondexcitation wavelength) and a fourth nucleotide type that lacks a labelthat is not, or minimally, detected in either channel (e.g. dGTP havingno label).

Further, as described in the incorporated materials of U.S. PatentApplication Publication No. 2013/0079232, sequencing data can beobtained using a single channel. In such so-called one-dye sequencingapproaches, the first nucleotide type is labeled but the label isremoved after the first image is generated, and the second nucleotidetype is labeled only after a first image is generated. The thirdnucleotide type retains its label in both the first and second images,and the fourth nucleotide type remains unlabeled in both images.

Some embodiments can utilize sequencing by ligation techniques. Suchtechniques utilize DNA ligase to incorporate oligonucleotides andidentify the incorporation of such oligonucleotides. Theoligonucleotides typically have different labels that are correlatedwith the identity of a particular nucleotide in a sequence to which theoligonucleotides hybridize. As with other SBS methods, images can beobtained following treatment of an array of nucleic acid features withthe labeled sequencing reagents. Each image will show nucleic acidfeatures that have incorporated labels of a particular type. Differentfeatures will be present or absent in the different images due thedifferent sequence content of each feature, but the relative position ofthe features will remain unchanged in the images. Images obtained fromligation-based sequencing methods can be stored, processed and analyzedas set forth herein. Exemplary SBS systems and methods which can beutilized with the methods and systems described herein are described inU.S. Pat. No. 6,969,488, U.S. Pat. No. 6,172,218, and U.S. Pat. No.6,306,597, the disclosures of which are incorporated herein by referencein their entireties.

Some embodiments can utilize nanopore sequencing (Deamer, D. W. &Akeson, M. “Nanopores and nucleic acids: prospects for ultrarapidsequencing.” Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D.Branton, “Characterization of nucleic acids by nanopore analysis”. Acc.Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin,and J. A. Golovchenko, “DNA molecules and configurations in asolid-state nanopore microscope” Nat. Mater. 2:611-615 (2003), thedisclosures of which are incorporated herein by reference in theirentireties). In such embodiments, the target nucleic acid passes througha nanopore. The nanopore can be a synthetic pore or biological membraneprotein, such as α-hemolysin. As the target nucleic acid passes throughthe nanopore, each base-pair can be identified by measuring fluctuationsin the electrical conductance of the pore. (U.S. Pat. No. 7,001,792;Soni, G. V. & Meller, “A. Progress toward ultrafast DNA sequencing usingsolid-state nanopores.” Clin. Chem. 53, 1996-2001 (2007); Healy, K.“Nanopore-based single-molecule DNA analysis.” Nanomed. 2, 459-481(2007); Cockroft, S. L., Chu, J., Amorin, M. & Ghadiri, M. R. “Asingle-molecule nanopore device detects DNA polymerase activity withsingle-nucleotide resolution.” J. Am. Chem. Soc. 130, 818-820 (2008),the disclosures of which are incorporated herein by reference in theirentireties). Data obtained from nanopore sequencing can be stored,processed and analyzed as set forth herein. In particular, the data canbe treated as an image in accordance with the exemplary treatment ofoptical images and other images that is set forth herein.

Some embodiments can utilize methods involving the real-time monitoringof DNA polymerase activity. Nucleotide incorporations can be detectedthrough fluorescence resonance energy transfer (FRET) interactionsbetween a fluorophore-bearing polymerase and γ-phosphate-labelednucleotides as described, for example, in U.S. Pat. No. 7,329,492 andU.S. Pat. No. 7,211,414 (each of which is incorporated herein byreference) or nucleotide incorporations can be detected with zero-modewaveguides as described, for example, in U.S. Pat. No. 7,315,019 (whichis incorporated herein by reference) and using fluorescent nucleotideanalogs and engineered polymerases as described, for example, in U.S.Pat. No. 7,405,281 and U.S. Patent Application Publication No.2008/0108082 (each of which is incorporated herein by reference). Theillumination can be restricted to a zeptoliter-scale volume around asurface-tethered polymerase such that incorporation of fluorescentlylabeled nucleotides can be observed with low background (Levene, M. J.et al. “Zero-mode waveguides for single-molecule analysis at highconcentrations.” Science 299, 682-686 (2003); Lundquist, P. M. et al.“Parallel confocal detection of single molecules in real time.” Opt.Lett. 33, 1026-1028 (2008); Korlach, J. et al. “Selective aluminumpassivation for targeted immobilization of single DNA polymerasemolecules in zero-mode waveguide nano structures.” Proc. Natl. Acad.Sci. USA 105, 1176-1181 (2008), the disclosures of which areincorporated herein by reference in their entireties). Images obtainedfrom such methods can be stored, processed and analyzed as set forthherein.

Some SBS embodiments include detection of a proton released uponincorporation of a nucleotide into an extension product. For example,sequencing based on detection of released protons can use an electricaldetector and associated techniques that are commercially available fromIon Torrent (Guilford, Conn., a Life Technologies subsidiary) orsequencing methods and systems described in US 2009/0026082 A1; US2009/0127589 A1; US 2010/0137143 A1; or US 2010/0282617 A1, each ofwhich is incorporated herein by reference. Methods set forth herein foramplifying target nucleic acids using kinetic exclusion can be readilyapplied to substrates used for detecting protons. More specifically,methods set forth herein can be used to produce clonal populations ofamplicons that are used to detect protons.

The above SBS methods can be advantageously carried out in multiplexformats such that multiple different target nucleic acids aremanipulated simultaneously. In particular embodiments, different targetnucleic acids can be treated in a common reaction vessel or on a surfaceof a particular substrate. This allows convenient delivery of sequencingreagents, removal of unreacted reagents and detection of incorporationevents in a multiplex manner. In embodiments using surface-bound targetnucleic acids, the target nucleic acids can be in an array format. In anarray format, the target nucleic acids can be typically bound to asurface in a spatially distinguishable manner. The target nucleic acidscan be bound by direct covalent attachment, attachment to a bead orother particle or binding to a polymerase or other molecule that isattached to the surface. The array can include a single copy of a targetnucleic acid at each site (also referred to as a feature) or multiplecopies having the same sequence can be present at each site or feature.Multiple copies can be produced by amplification methods such as, bridgeamplification or emulsion PCR as described in further detail below.

The methods set forth herein can use arrays having features at any of avariety of densities including, for example, at least about 10features/cm², 100 features/cm², 500 features/cm², 1,000 features/cm²,5,000 features/cm², 10,000 features/cm², 50,000 features/cm², 100,000features/cm², 1,000,000 features/cm², 5,000,000 features/cm², or higher.

An advantage of the methods set forth herein is that they provide forrapid and efficient detection of a plurality of target nucleic acid inparallel. Accordingly the present disclosure provides integrated systemscapable of preparing and detecting nucleic acids using techniques knownin the art such as those exemplified above. Thus, an integrated systemof the present disclosure can include fluidic components capable ofdelivering amplification reagents and/or sequencing reagents to one ormore immobilized DNA fragments, the system comprising components such aspumps, valves, reservoirs, fluidic lines and the like. A flow cell canbe configured and/or used in an integrated system for detection oftarget nucleic acids. Exemplary flow cells are described, for example,in US 2010/0111768 A1 and U.S. Ser. No. 13/273,666, each of which isincorporated herein by reference. As exemplified for flow cells, one ormore of the fluidic components of an integrated system can be used foran amplification method and for a detection method. Taking a nucleicacid sequencing embodiment as an example, one or more of the fluidiccomponents of an integrated system can be used for an amplificationmethod set forth herein and for the delivery of sequencing reagents in asequencing method such as those exemplified above. Alternatively, anintegrated system can include separate fluidic systems to carry outamplification methods and to carry out detection methods. Examples ofintegrated sequencing systems that are capable of creating amplifiednucleic acids and also determining the sequence of the nucleic acidsinclude, without limitation, the MiSeq™ platform (Illumina, Inc., SanDiego, Calif.) and devices described in U.S. Ser. No. 13/273,666, whichis incorporated herein by reference.

In the methods and compositions presented herein, polynucleotides areimmobilized to the solid support. In some embodiments, thepolynucleotides are covalently immobilized to the support. Whenreferring to immobilization of molecules (e.g. nucleic acids) to a solidsupport, the terms “immobilized” and “attached” are used interchangeablyherein and both terms are intended to encompass direct or indirect,covalent or non-covalent attachment, unless indicated otherwise, eitherexplicitly or by context. In certain embodiments of the inventioncovalent attachment may be preferred, but generally all that is requiredis that the molecules (e.g. nucleic acids) remain immobilized orattached to the support under the conditions in which it is intended touse the support, for example in applications requiring nucleic acidamplification and/or sequencing.

Certain embodiments of the invention may make use of solid supportscomprised of an inert substrate or matrix (e.g. glass slides, polymerbeads etc.) which has been functionalized, for example by application ofa layer or coating of an intermediate material comprising reactivegroups which permit covalent attachment to biomolecules, such aspolynucleotides. Examples of such supports include, but are not limitedto, polyacrylamide hydrogels supported on an inert substrate such asglass, particularly polyacrylamide hydrogels as described in WO2005/065814 and US 2008/0280773, the contents of which are incorporatedherein in their entirety by reference. In such embodiments, thebiomolecules (e.g. polynucleotides) may be directly covalently attachedto the intermediate material (e.g. the hydrogel) but the intermediatematerial may itself be non-covalently attached to the substrate ormatrix (e.g. the glass substrate). The term “covalent attachment to asolid support” is to be interpreted accordingly as encompassing thistype of arrangement.

Exemplary covalent linkages include, for example, those that result fromthe use of click chemistry techniques. Exemplary non-covalent linkagesinclude, but are not limited to, non-specific interactions (e.g.hydrogen bonding, ionic bonding, van der Waals interactions etc.) orspecific interactions (e.g. affinity interactions, receptor-ligandinteractions, antibody-epitope interactions, avidin-biotin interactions,streptavidin-biotin interactions, lectin-carbohydrate interactions,etc.). Exemplary linkages are set forth in U.S. Pat. Nos. 6,737,236;7,259,258; 7,375,234 and 7,427,678; and US Pat. Pub. No. 2011/0059865A1, each of which is incorporated herein by reference.

The terms “target nucleic acid,” “target nucleic acid molecule,” “targetnucleic acid species” and any grammatical equivalent thereof, refer tonucleic acid molecules that are desired to be detected, sequenced orotherwise analyzed. Any of a variety of desired target nucleic acidmolecules can be utilized, including but not limited to exons, ornucleic acid molecules complementary thereto; cDNA molecules, or nucleicacid molecules complementary thereto; untranslated regions (UTRs) ornucleic acids complementary thereto; promoter and/or enhancer regions,or nucleic acid molecules complementary thereto; evolutionary conservedregions (ECRs), or nucleic acid molecules complementary thereto;transcribed genomic regions, or nucleic acid molecules complementarythereto. Any of a variety of methods can be used to obtain targetednucleic acid molecules, as disclosed herein. Such methods include, butare not limited to, obtaining a targeted nucleic acid molecule usinghybridization-extension capture enrichment; using targeted restrictionsites, for example, using an oligonucleotide engineered with a hairpinhaving a Type IIS restriction enzyme site such as a Fold restrictionenzyme site and a locus-specific region; using locus-specifichyperbranched rolling circle amplification; using random-locus-specificprimer amplification; using multiplex emulsion PCR; using multiplexbridge PCR; using padlock probe amplification; and using mini-librariesfrom targeted libraries, as disclosed herein.

As used herein, the terms “target nucleic acid sequence,” “samplenucleic acid sequence” and like terms refer to nucleic acid sequencesobtained from samples that are desired to be analyzed.

A nucleic acid sample that is amplified, sequenced or otherwisemanipulated in a method disclosed herein can be, for example, DNA orRNA. Exemplary DNA species include, but are not limited to, genomic DNA(gDNA), mitochondrial DNA, chloroplast DNA, episomal DNA, viral DNA andcopy DNA (cDNA). One non-limiting example of a subset of genomic DNA isone particular chromosome or one region of a particular chromosome.Exemplary RNA species include, without limitation, coding RNA such asmessenger RNA (mRNA), and non-coding RNA (ncRNA) such as transfer RNA(tRNA), microRNA (miRNA), small nuclear RNA (snRNA) and ribosomal RNA(rRNA). Further species of DNA or RNA include fragments or portions ofthe species listed above or amplified products derived from thesespecies, fragments thereof or portions thereof. The methods describedherein are applicable to the above species encompassing all or part ofthe complement present in a cell. For example, using methods describedherein the sequence of a substantially complete genome can be determinedor the sequence of a substantially complete targeted nucleic acidsequences such as mRNA or cDNA complement of a cell can be determined.

Target DNA molecules of different sequence may be prepared by mixing anumber, greater than one, of individual DNA molecules. In the preferredprocedure, genomic DNA is fragmented into small molecules, preferablyless than 1000 base pairs in length. Fragmentation of DNA may beachieved by a number of methods including: enzymatic digestion, chemicalcleavage, sonication, nebulisation, or hydroshearing, preferablynebulization.

Known sequences may be added to the 3′ and 5′ ends of target nucleicacid sequences using any of a number of methodologies known in the art,as exemplified, for example by those set forth in U.S. Pat. Nos.7,741,463, 7,985,565, and 7,115,400, each of which is incorporated byreference herein in its entirety. For example, in some embodiments,known sequences are added by ligating adapters to nucleic acidfragments. In some embodiments, known sequences are added byamplification using primers having additional known sequence at their 5′ends. In some embodiments, known sequences can comprise sequences thatare identical or complimentary to amplification primers immobilized on asolid support. For example, in certain embodiments, known sequence caninclude either one of two universal capture regions, such as P5 or P7regions. A P5 region includes the nucleotide sequence5′-AATGATACGGCGACCACCGA-3′. A P7 region includes the nucleotide sequence5′-CAAGCAGAAGACGGCATACGA-3′. In certain embodiments, the known sequenceis the reverse complement of the P5 region sequence (“anti-P5”:5′-TCGGTGGTCGCCGTATCATT-3′) or the P7 region sequence (“anti-P7”:5′-TCGTATGCCGTCTTCTGCTTG-3′) amplification primer. In certainembodiments, the oligonucleotide can hybridize with Illumina®amplification primers P5 (paired end)(5′-AATGATACGGCGACCACCGAGAUCTACAC-3′) or P7 (paired end)(5′-CAAGCAGAAGACGGCATACGA(8-oxo-G)AT-3′). In certain embodiments, theoligonucleotide can hybridize with the reverse complement of theIllumina® capture primer P5 (paired end) (“anti-P5 (paired end)”:5′-GTGTAGATCTCGGTGGTCGCCGTATCATT-3′) or P7 (paired end) (“anti-P7(paired end)”: 5 ‘-ATCTCGTATGCCGTCTTCTGCTTG-3’).

The terms “solid surface,” “solid support” and other grammaticalequivalents herein refer to any material that is appropriate for or canbe modified to be appropriate for the attachment of the transposomecomplexes. As will be appreciated by those in the art, the number ofpossible substrates is very large. Possible substrates include, but arenot limited to, glass and modified or functionalized glass, plastics(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes,Teflon™, etc.), polysaccharides, nylon or nitrocellulose, ceramics,resins, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, plastics, optical fiberbundles, and a variety of other polymers. Particularly useful solidsupports and solid surfaces for some embodiments are located within aflow cell apparatus. Exemplary flow cells are set forth in furtherdetail below.

In some embodiments, the solid support comprises a patterned surfacesuitable for immobilization of transposome complexes in an orderedpattern. A “patterned surface” refers to an arrangement of differentregions in or on an exposed layer of a solid support. For example, oneor more of the regions can be features where one or more transposomecomplexes are present. The features can be separated by interstitialregions where transposome complexes are not present. In someembodiments, the pattern can be an x-y format of features that are inrows and columns. In some embodiments, the pattern can be a repeatingarrangement of features and/or interstitial regions. In someembodiments, the pattern can be a random arrangement of features and/orinterstitial regions. In some embodiments, the transposome complexes arerandomly distributed upon the solid support. In some embodiments, thetransposome complexes are distributed on a patterned surface. Exemplarypatterned surfaces that can be used in the methods and compositions setforth herein are described in U.S. Ser. No. 13/661,524 or US Pat. App.Publ. No. 2012/0316086 A1, each of which is incorporated herein byreference.

In some embodiments, the solid support comprises an array of wells ordepressions in a surface. This may be fabricated as is generally knownin the art using a variety of techniques, including, but not limited to,photolithography, stamping techniques, molding techniques andmicroetching techniques. As will be appreciated by those in the art, thetechnique used will depend on the composition and shape of the arraysubstrate.

The composition and geometry of the solid support can vary with its use.In some embodiments, the solid support is a planar structure such as aslide, chip, microchip and/or array. As such, the surface of a substratecan be in the form of a planar layer. In some embodiments, the solidsupport comprises one or more surfaces of a flowcell. The term“flowcell” as used herein refers to a chamber comprising a solid surfaceacross which one or more fluid reagents can be flowed. Examples offlowcells and related fluidic systems and detection platforms that canbe readily used in the methods of the present disclosure are described,for example, in Bentley et al., Nature 456:53-59 (2008), WO 04/018497;U.S. Pat. No. 7,057,026; WO 91/06678; WO 07/123744; U.S. Pat. No.7,329,492; U.S. Pat. No. 7,211,414; U.S. Pat. No. 7,315,019; U.S. Pat.No. 7,405,281, and US 2008/0108082, each of which is incorporated hereinby reference.

In some embodiments, the solid support or its surface is non-planar,such as the inner or outer surface of a tube or vessel. In someembodiments, the solid support comprises microspheres or beads. By“microspheres” or “beads” or “particles” or grammatical equivalentsherein is meant small discrete particles. Suitable bead compositionsinclude, but are not limited to, plastics, ceramics, glass, polystyrene,methylstyrene, acrylic polymers, paramagnetic materials, thoria sol,carbon graphite, titanium dioxide, latex or cross-linked dextrans suchas Sepharose, cellulose, nylon, cross-linked micelles and teflon, aswell as any other materials outlined herein for solid supports may allbe used. “Microsphere Detection Guide” from Bangs Laboratories, FishersInd. is a helpful guide. In certain embodiments, the microspheres aremagnetic microspheres or beads.

The beads need not be spherical; irregular particles may be used.Alternatively or additionally, the beads may be porous. The bead sizesrange from nanometers, i.e. 100 nm, to millimeters, i.e. 1 mm, withbeads from about 0.2 micron to about 200 microns being preferred, andfrom about 0.5 to about 5 micron being particularly preferred, althoughin some embodiments smaller or larger beads may be used.

Example 1

This example describes solid phase amplification according to oneembodiment, as illustrated in FIG. 1. A standard universal PhiX or CT418library containing P5 and P7′ regions was used to attach the additionalP5′ at the 5′ end of the already prepared library. This was accomplishedby performing additional 18 PCR cycles. The libraries were diluted to200 pM final and amplified using either standard primer or P7-P5′primer. Each 50 ul PCR reaction contained 22 μl of H2O, 25 μl of 2×Phusion Mastermix (NEB), 1 μl each of the appropriate primer and DNA.After PCR, the resulting library concentrations were determined by theNanodrop (Thermo Scientific) and diluted to 10 nM in buffer EB(QIAGEN)+0.05% Tween20. A flowcell was prepared by grafting HEG primers(lanes 1-4) or standard PE primers (lanes 5-8) using the protocoldescribed in U.S. Pat. Nos. 8,536,477, 8,715,966, and U.S. PatentApplication Pub. 2008/0280773, the content of each of which isincorporated by reference herein in its entirety. The librariesgenerated above were used to generate clusters on the grafted flowcell.

The clusters were amplified using TruSeq cluster generation kit forGenome Analyzer (Illumina) as per manufacturer's recommended protocolfor 35 cycles. The flow cell was stained with SYBR Green (MolecularProbes) and imaged on a fluorescence microscope. As shown in the toppanel of FIG. 4A, the clusters generated with standard primers resultedin normal clusters whereas the same with extra P5 modified primers wereshown to result in a mix of large and normal clusters (FIG. 4A bottompanel). The plots of number of clusters versus the SYBR signal intensityfrom the flowcells described in FIG. 4A are shown in FIG. 4B. Theclusters from standard primers resulted in low SYBR intensity (topgraph) whereas those from modified primer with added P5 resulted in 10times higher intensity (bottom graph).

The flowcell was then prepared to do a first cycle of sequencingincorporation, using a white light fluorophore set. The first cycleimages of C, T and G with the grayscale of the image adjusted to thebrightness of the T image on the modified primer lane showed that theclusters using the modified primers are brighter than the clusters madeon standard primers. (FIG. 5)

Example 2

This example describes solid phase amplification according to anembodiment, as illustrated in FIG. 3. The experiments reported in FIG. 6show the results from concatemerization of 20T/A linkers on the P5/P7grafting primers (referred as 20T-P7/20A-P5) instead of attaching theconcatamer facilitating primers on to the library during PCR process. Inthis experiment, the flowcell was grafted with 20A/T Paired-end P5/P7primers alongside standard primers. The standard grafting protocol asdescribed in incorporated materials of U.S. Pat. Nos. 8,536,477,8,715,966, and U.S. Patent Application Pub. 2008/0280773 was followedand this grafted flowcell was used for cluster amplification on a cBOTclonal amplification system (Illumina Cat # SY-301-2002) using theTruSeq cluster generation kit for Genome Analyzer (Illumina) as permanufacturer's recommended protocol. CT418 libraries were used astemplate; amplified 105 cycles at 60° C. The flowcell was stained withSYBR Green and imaged as described in FIG. 4. The plots of number ofclusters versus SYBR signal intensity showed that using 20T-P7/20A-P5surface primers (6A) the cluster brightness increased compared to thestandard surface primers (6B). However, fewer clusters with higherintensity were observed as compared to the example given in FIGS. 4 and5.

Effect of Temperature on Cluster Intensity:

Temperature ranges from 50° to 60° C. and amplification cycles rangingfrom 25 to 45 were tested for cluster generation. The grafting offlowcell and the experiment procedure were as described above for FIG. 6except temperatures of 50 and 55° C. were tested for the clusteramplification for 35 or 45 cycles. As shown in FIG. 7, the 20T/20Asurface primers amplified at 50° C. and stained with SYBR Green showedthat 20T/20A clusters are 3-5 times brighter than those with standardsurface primers (7A). The cluster diameter remained the same for 20T/20Asurface primers and standard primers (7B). The libraries used were 1, 9and 18 pM PhiX and 1 pM CT418. The inventors found that at temperaturesat or below 55° C. and 45-cycle amplification resulted in brighterclusters using 20T/20A primers compared to standard primers. Increasingthe Bst polymerase concentration along with increased extension times to72 sec from standard 36 sec used during bridge amplification alsoresulted in brighter clusters when 20T/20A primers were used compared tostandard clusters (data not shown).

FIG. 8 is a side-by-side comparison of cluster intensity over cyclenumber during a standard 100 cycle sequencing run on a GA, showing thatthe intensity of 20T-P7/20A-P5 primer clusters at the end of a 100 cyclerun is similar to the intensity of clusters with standard primers at thestart of the run.

Throughout this application various publications, patents and/or patentapplications have been referenced. The disclosure of these publicationsin their entireties is hereby incorporated by reference in thisapplication.

The term comprising is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the following claims.

1. A method of preparing immobilized templates for a nucleic acidsequencing reaction comprising: (a) providing a solid support having aforward and reverse amplification primer immobilized thereon; (b)providing a target nucleic acid, wherein the target nucleic acidcomprises: (i) a first region of known sequence complementary to theforward amplification primer; (ii) a first template region; (iii) asecond region of known sequence substantially identical to the reverseamplification primer, wherein the first template region is between thefirst region of known sequence and the second region of known sequence;and (iv) a third region of known sequence complementary to the forwardamplification primer, wherein the first template region and the secondregion of known sequence are between the first region of known sequenceand the third region of known sequence; (c) applying the target nucleicacid to the solid support under conditions suitable for hybridizationwhereby the first region of known sequence hybridizes to the forwardamplification primer; and (d) extending the hybridized forwardamplification primer to generate an immobilized template comprising acomplementary copy of the target nucleic acid.
 2. The method of claim 1,further comprising: (e) denaturing the target nucleic acid from theimmobilized template; (f) hybridizing the immobilized template to thereverse amplification primer, whereby a complementary copy of the secondregion of known sequence hybridizes to the reverse amplification primer;and (g) extending the hybridized reverse amplification primer togenerate a second immobilized strand comprising the first templateregion positioned between the first region of known sequence and thesecond region of known sequence.
 3. The method of claim 2, furthercomprising: (h) denaturing the second immobilized strand from the firstimmobilized template; (i) hybridizing the second immobilized strand tothe first immobilized template, whereby the first region of knownsequence of the second immobilized strand hybridizes to a complementarycopy of the first region of known sequence in the immobilized template;(j) extending a 3′ OH of the first region of known sequence to generatea concatemer of the second immobilized strand; and (k) extending a 3′ OHof the complementary copy of the first region of known sequence togenerate a concatemer of the first immobilized template.
 4. The methodof claim 3, further comprising: (l) denaturing the concatemers andrepeating steps (i), (j) and (k) to generate further concatemers of eachstrand.
 5. The method of claim 1, wherein the forward amplificationprimer comprises a forward complementarity region, said forwardcomplementarity region having complementarity to a reversecomplementarity region of the reverse amplification primer.
 6. Themethod of claim 5, wherein the forward complementarity region ispositioned directly 5′ of a region having complementarity to the firstregion of known sequence of the target nucleic acid.
 7. The method ofclaim 5, wherein the reverse complementarity region is positioneddirectly 5′ of a region having sequence substantially identical to thesecond region of known sequence of the target nucleic acid.
 8. Themethod of claim 5, wherein the forward complementarity region and secondcomplementarity region are configured such that repeated cycles ofhybridization and extension produces a concatemer of the firstimmobilized strand, each copy in the concatemer separated from the nextby sequence from the forward complementarity region.
 9. The method ofclaim 5, wherein the forward complementarity region and secondcomplementarity region are configured such that repeated cycles ofhybridization and extension produces a concatemer of the secondimmobilized strand, each copy in the concatemer separated from the nextby sequence from the reverse complementarity region.
 10. The method ofclaim 3, wherein said concatemers comprises multiple copies of saidtarget nucleic acid molecule of at least 10, 20, 50, 100, 200, 500,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or at least 10,000copies.
 11. The method of claim 1, wherein said solid support is planar.12. The method of claim 1, wherein said solid support comprisesmicrowells.
 13. The method of claim 1, wherein said target nucleic acidhas a length of at least 10, 20, 50, 100, 200 or at least 500nucleotides.
 14. The method of claim 1, wherein said target nucleic acidfurther comprises a fourth region of known sequence substantiallyidentical to the reverse amplification primer, wherein the first regionof known sequence, the first template region and the second region ofknown sequence are between the third region of known sequence and thefourth region of known sequence.
 15. The method of claim 1, whereineither of the forward amplification primer and the reverse amplificationprimer comprises a non-nucleotide chemical linker moiety positioned toprevent copying of any nucleotides that are 5′ of the non-nucleotidechemical linker moiety.
 16. (canceled)
 17. The method of claim 15,wherein the non-nucleotide chemical linker moiety comprises a diol. 18.The method of claim 15, wherein the non-nucleotide chemical linkermoiety comprises a non-nucleotide linker tethering the primer to thesolid support.
 19. The method of claim 3, further comprising sequencingthe target nucleic acid.
 20. The method of claim 19, wherein the step ofsequencing the target nucleic acid comprises: hybridizing one or moresequencing primers to the first immobilized template or the secondimmobilized strand; extending the sequencing primers by incorporatingone or more labeled nucleotides into the nascent strand; and detectingthe labeled nucleotides, thereby obtaining sequence information aboutthe target nucleic acid.
 21. The method of claim 1, wherein the targetnucleic acid is prepared by amplifying with a pair of primers comprisinga binding moiety whereby one strand of an amplification productgenerated using the pair of primers can be separated from the oppositestrand.
 22. (canceled)